Toxicity and medical countermeasure

studies on nerve agents VX and VM

Helen Rice, Chris Dalton, Matt Price, Stuart Graham, Christopher Green, John Jenner, Helen Groombridge and Christopher Timperley

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Nerve agents of Syrian Arab Republic

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Synthesis of 14C-labelled VM and VX

Nerve agents of Syrian Arab Republic

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C M Timperley et al., Med. Chem. Commun. 2012, 3, 352-356

AChE

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C M Timperley et al., Med. Chem. Commun. 2012, 3, 352-356

AChE

Nerve agent poisoning VX Human LD50 No treatment

Photo: Ed Clarkson, USAMRICD Therapy

Inhaled volatile agent e.g. sarin

All Agent Absorbed

SIGNS Minutes

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Persistent liquid agent on skin e.g. VX

Minutes Hours

Agent continues to be absorbed

SIGNS

Therapy

PK & PD for percutaneous VX

0.0

0.5

1.0

1.5

ChE

act

ivity

(µm

ol/m

in/m

L)

0 12 24 36 480.00

0.05

0.10

Time (hours)

VX c

once

ntra

tion

(ng/

mL)

n = 7

Red Cell AChE activity

VX brain concentration

VX muscle concentration

Plasma BChE activity

H Mumford et al., Chem. Bio. Interact., 2013, 203, 160

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~0.5 × LD50 VX

Measurement of agent diffusion in vitro

• Franz type diffusion cell • application volume 10 µL • area 2.54 cm² • temperature 32 °C • receptor volume ~5 mL • stirred continuously

Donor Chamber

Sampling arm

Cap

Skin

Receptor chamber

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Guinea-pig skin No significant difference between combined penetration rates of either neat or dilute agent mixtures was observed

0 3 6 9 12 15 18 21 240.0000.0050.0100.0150.0200.0250.0300.0350.0400.0450.050

VX 100:VM 0VX 75:VM 25VX 50:VM 50VX 25:VM 75VX 0:VM 100

Time (h)

Amou

nt P

enet

rate

d (m

g.cm

-2)

0 3 6 9 12 15 18 21 240.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

VX 100:VM 0VX 75:VM 25VX 50:VM 50VX 25:VM 75VX 0:VM 100

Time (h)

Amou

nt P

enet

rate

d (m

g.cm

-2) Neat agent Dilute agent

H Rice et al., Proc Roy Soc A. 2015, 471, 20140891

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Pig skin

0 3 6 9 12 15 18 21 240.0

0.2

0.4

0.6

0.8

1.0

VX 100:VM 0VX 75:VM 25VX 50:VM 50VX 25:VM 75VX 0:VM 100

Time (h)

Amou

nt P

enet

rate

d (m

g.cm

-2)

0 3 6 9 12 15 18 21 240.000

0.002

0.004

0.006

0.008

0.010

0.012

VX 100:VM 0VX 75:VM 25VX 50:VM 50VX 25 :VM 75VX 0:VM 100

Time (h)

Amou

nt P

enet

rate

d (m

g.cm

- 2)Neat agent Dilute agent

No significant difference between combined penetration rates of either neat or dilute agent mixtures was observed

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H Rice et al., Proc Roy Soc A. 2015, 471, 20140891

Historical results comparison

Penetration of 14C VX µg.cm-2.h-1 (mean ± SD)

Guinea-pig skin Pig skin Human skin

Previous study * 3.69 ± 0.72 0.73 ± 0.35 1.01 ± 0.21

Current study 5.23 ± 0.95 0.74 ± 0.43 n.d.

* C Dalton et al., Toxicology in vitro 2006, 20, 1532

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n.d. = not determined

Experimental design: in vivo Dunkin-Hartley guinea-pigs: male, conscious

Toxicity - 24 h percutaneous LD50 – VM alone and VM-VX mixtures (3 different proportions) – agent diluted in isopropanol and applied to clipped skin – left unoccluded and no decontamination

MedCM study - 2 × LD50 challenge – VM 2.388 mg·kg -1 or VX 1.226 mg·kg -1, – Therapy (i.m.) on signs of cholinergic poisoning and

worsening signs of poisoning (max. 3 doses over 9 h)

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Results: 24 h LD50

0 20 40 60 80 100

0

500

1000

1500

2000

LD50 value (95% CI)Predicted (95% CI)

y=100/((x/TA))+((100-x)/TB))

TA = Toxicity (LD50) of ATB = Toxicity (LD50) of B

VX% in Mixture (balance VM)

Pred

icte

d/M

easu

red

LD50

( µg/

kg)

LD50 VX alone 0.613 mg.kg-1

LD50 VM alone 1.290 mg.kg-1

H Rice et al., Proc Roy Soc A. 2015, 471, 20140891

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0 2 4 6 80

50

100

24

VM:HI-6VM:P2SVM:SalineVX:HI-6VX:P2SVX:Saline

o/nTime (hours)

Perc

ent s

urvi

val

Therapy (3 ×) of atropine, avizafone and either P2S or HI-6 did not protect guinea pigs from 2 × LD50 of either VM or VX • Therapy on signs of poisoning and worsening signs of poisoning

prolonged the times to death compared to saline-treated controls • At 6 h, survival was significantly higher in treated groups than saline

controls, for both nerve agents and either treatment

Therapy oxime 30.0 mg.kg-1 atropine 17.4 mg.kg-1 avizafone 3.14 mg.kg-1

oxime = P2S or HI6 DMS

H Rice et al., Proc. Roy. Soc. A. 2015, 471, 20140891

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Conclusions VM is approximately half as toxic as VX by the p.c. route

In-service (P2S) or future oxime (HI-6) MedCM did not fully protect guinea-pigs but extended time to death to > 5-6 h

Mixtures of VM + VX do not penetrate skin faster than the individual agent

Mixtures of VM + VX were not more toxic than the pure agent

There is no requirement to handle mixtures of VM + VX in demilitarisation operations differently from the pure materials

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MedCM are part of a system of protection

Effective, acceptable, practicable and affordable

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Publications

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Acknowledgements

• Staff of Biology Group

• Analytical Chemistry Team

• Synthetic Chemistry Team

• Veterinary Surgeon

• Animal care staff

• Work carried out under a project licence issued by UK Home Office under the Animals (Scientific Procedures) Act 1987

• Work was funded by the UK MOD

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